1. Field of the Invention
This invention relates to compositions and methods for controlling bacterial growth, and more particularly to anti-bacterial agents and their methods of use.
2. Description of the Related Art
Bacterial infections remain a public health concern, and indeed a growing one in view of increasing resistance to existing drugs by pathogenic bacteria. Drugs now in use fall into a relatively small number of chemical classes, and operate by one of a relatively small number of mechanisms. Development of resistance to one drug can therefore adversely affect the efficacy of others. Moreover, because bacteria can exchange genetic information, resistance can spread from one species to another.
It is therefore desirable to have new classes of anti-bacterial agents based upon novel mechanisms of action, to which bacteria are unlikely to have resistance.
In addition, bacteria in nature commonly grow attached on solid surfaces in a mode of growth referred to as a biofilm. Bacteria within biofilms differ physiologically from those grown in liquid culture (planktonic cells) in having increased resistance to environmental stresses (such as antibiotic treatment). In clinical environments, biofilms of pathogenic bacteria lead to persistent and chronic infections refractory to treatment with conventional antibiotics. The U.S. Centers for Disease Control estimate that 60% of bacterial infections involve such biofilms. Industrially, biofilms contaminate and clog water lines, foul surfaces and contribute to corrosion and decay. Not all the consequences of biofilm formation are deleterious, however; for example, in bioproduction processes biofilms help in maintaining a stable population of cells as substrate passes through a bioreactor.
Consequently it is desirable not only to have new classes of anti-bacterial agents, but also to have ways of promoting and/or inhibiting the formation and maintenance of bacterial biofilms.
A preferred embodiment provides a method of controlling bacterial growth, comprising exposing a bacterium to a compound of structure I 
wherein E is selected from the group consisting of B, P, and S, T1, and T2 are each independently selected from the group consisting of O, NR, and CH2, where R=H or C1-C8 alkyl, or C1-C8 oxoalkyl, and L is selected from the group consisting of ethylene, propylene, and four to six-membered alicyclic and aromatic rings, provided that structure I does not include AI-2-borate. Preferably, E is B (boron) or P (phosphorous). Preferably, T1, and T2 are O (oxygen). Preferably, the compound has a molecular weight less than about 750 Da, more preferably, less than about 500 Da. In preferred embodiments, bacterial growth is controlled by administering a therapeutically effective amount of the compound to a human infected with the bacterium.
Another preferred embodiment provides a pharmaceutical composition comprising a compound having structure I 
wherein E is selected from the group consisting of B, P, and S, T1 and T2 are each independently selected from the group consisting of O, NR, and CH2, where R=H or C1-C8 alkyl, or C1-C8 oxoalkyl, and L is selected from the group consisting of ethylene, propylene, and four to six-membered alicyclic and aromatic rings, provided that structure I does not include AI-2-borate. Preferably, L is tetrahydrofuran bearing a keto, a hydroxy, and a carboxamido functional group, T1 and T2 are oxygen, and E is B or P. More preferably, the compound has the following structure: 
These and other embodiment are described in greater detail below.
Preferred embodiments provide pharmaceutical compositions comprising compounds having the structure I: 
wherein E is selected from the group consisting of B, P, and S, T1, and T2 are each independently selected from the group consisting of O, NR, and CH2, where R=H or C1-C8 alkyl, or C1-C8 oxoalkyl, and L is selected from the group consisting of ethylene, propylene, and four to six-membered alicyclic and aromatic rings, provided that structure I does not include AI-2-borate.
Preferred L groups include ethylene, propylene, cyclopentyl, cyclohexyl, pyrrolidine, tetrahydrofuran, piperidine, pyran, dioxane, morpholine, pyrrole, furan, pyridine, pyrimidine, pyrazine, imidazole, thiazole, oxazole, purine, and indazole. Particularly preferred L groups include ethylene, propylene, cyclopentyl, cyclohexyl, pyrrolidine, tetra-hydrofuran, piperidine, pyran, dioxane, and morpholine. Most preferred L groups include cyclopentyl, cyclohexyl, pyrrolidine, tetrahydrofuran, piperidine, pyran, dioxane, and morpholine. A particularly preferred compound has the structure II, where E is B or P: 
Other specific examples of preferred compounds include cyclic borate, sulfate, or phosphate esters and amides, as shown below for compounds derived from [3.3.0] bicyclooctane: 
The unsubstituted five-membered ring in the above structures can incorporate one or more heteroatoms, with hydrogen-bonding heteroatoms such as O and N being particularly preferred. The unsubstituted five-membered ring in the above structures can also bear one or more substituents containing heteroatoms, again with hydrogen-bonding heteroatoms such as O and N being particularly preferred.
Additional preferred embodiments include compounds derived from [3.4.0] bicyclononane. Specific examples include the following: 
Compounds having hydrogen-bonding substituents at a bridgehead position are also particularly preferred, in view of the strong H-bonding to this position evident in the structure found for the luxP-AI-2 co-crystal (discussed below). Examples of such compounds include the following, with R=OH or NH2 being particularly preferred: 
Methods of making various specific compounds are described in the Examples below. Methods of making the other compounds described herein may be undertaken by modifying the syntheses described in the Examples below in a manner known to those skilled in the art.
Specific compounds for a particular application are preferably selected with the aid of computer-aided drug design and/or combinatorial chemistry methods that are well-known to those skilled in the art. Computers and software suitable for carrying out computer-aided drug design are commercially available. Preferred computer packages include Sybyl version 6.8 from Tripos, Inc. and MacroModel version 8.0 from Schrodinger Software. Preferably, the heteroatoms are spatially disposed to interact with hydrogen-bonding groups on the LuxP protein.
One application of the present invention is in influencing the development or maintenance of biofilms, communities of bacteria that grow attached to solid surfaces. Bacteria within biofilms often exhibit greater resistance to antibiotic treatment than those living freely, and hence commonly lead to persistent and chronic infections refractory to treatment. The U.S. Centers for Disease Control estimate that 60% of bacterial infections involve such biofilms. Industrially, biofilms contaminate and clog water lines, foul surfaces and contribute to corrosion and decay. Not all the consequences of biofilm formation are deleterious, however; for example, in bioproduction processes biofilms help in maintaining a stable population of cells as substrate passes through a bioreactor.
Quorum-sensing influences biofilm formation, and therefore ways of promoting or impeding quorum-sensing also provide ways of controlling biofilm formation, including biofilm growth. For example, compounds of structure I can be used to affect biofilms by either stimulating their formation or hindering it. Methods for promoting or impeding biofilm formation are preferably practiced by exposing the bacteria to the compound in an amount that affects biofilm formation. Particular amounts for a given application may be determined by routine experimentation in a manner generally known to those skilled in the art.
Reference to a particular compound herein is to be understood as a reference to the compound itself and any salts thereof, and vice versa. Compounds that possess an acidic or basic group may form pharmaceutically-acceptable salts with pharmaceutically-acceptable cations or anions. Examples of pharmaceutically-acceptable cations include ammonium, tetramethylammonium, alkali metal (e.g. sodium, lithium and potassium) and alkaline earth metal (e.g. calcium, barium and magnesium), aluminum, zinc, and bismuth cations, and protonated forms of basic amino acids, such as arginine, lysine, and organic amines such as ethanolamine, ethylenediamine, triethanoleamine, benzylphenethylamine, methylamine, dimethylamine, trimethylamine, diethylamine, piperidine, morpholine, tris-(2-hydroxyethyl)amine, and piperazine.
Examples of pharmaceutically-acceptable anions include those derived from inorganic acids such as hydrochloric, hydrobromic, hydriodic, sulfuric, and phosphoric acid, as well as organic acids such as p-toluenesulfonic, methanesulfonic, oxalic, p-bromo-phenylsulfonic, carbonic, succinic, citric, benzoic, and acetic acid, and related inorganic and organic acids. Such pharmaceutically-acceptable salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, ammonium, monohydrogenphosphate, dihydrogenphosphate, meta-phosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, hippurate, butyne-1,4-dioate, hexane-1,6-diospate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, xcex1-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate. It is understood that the above salts may form hydrates or exist in a substantially anhydrous form.
The compounds described herein may be administered directly to subjects, preferably humans, and/or may be administered in the form of pharmaceutical compositions comprising one or more of the compounds. A preferred mode of administration of the compound is oral. Oral compositions preferably include an inert diluent and/or an edible carrier. The compound can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and/or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents. The compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the compound, sucrose as a sweetening agent and preservatives, dyes and colorings and flavors.
The compound can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics. Preferred antibiotics for this purpose include aminoglycosides such as tobramycin, glycopeptides such as vancomycin, beta lactams such as amoxicillin, quinolones such as ciprofloxicin, macrolides such as azithromycin, tetracyclines, sulfonamides, trimethoprim-sulfamethoxazole, or chloramphenicol. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. If administered intravenously, preferred carriers are physiological saline or phosphate buffered saline (PBS).
In a preferred embodiment, the compound is prepared with carriers that protect it against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are known to those skilled in the art.
Pharmaceutical compositions are preferably administered to subjects, preferably humans, in an amount that is therapeutically effective to treat a bacterial infection. Therapeutically effective amounts can be determined by those skilled in the art by such methods as clinical trials. Dosage may be adjusted in individual cases as required to achieve the desired degree of target bacterial regulation. Sustained release dosages and infusions are specifically contemplated. Pharmaceutical compositions can be administered by any appropriate route for systemic, local or topical delivery, for example, orally, parenterally, intravenously, intradermally, subcutaneously, buccally, intranasally, by inhalation, vaginally, rectally or topically, in liquid or solid form. Methods of administering the compounds described herein may be by specific dose or by controlled release vehicles.
The pharmaceutical composition may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compound, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed methods.
In a particular case, the therapeutically effective amount of a pharmaceutical composition to be used in the treatment of a bacterial infection will typically vary with the severity of the infection and the route by which the drug is administered. The dose, and perhaps the dose frequency, will also vary according to the age, body weight, and response of the individual patient. In general, the total daily dose range of the present compounds for a 70 kg person is from about 1 mg to about 2000 mg, in single or divided doses. Preferably, a daily dose range for a 70 kg person should be between about 5 mg and about 1500 mg, in single or divided doses. More preferably, a daily dose range for a 70 kg person should be between about 10 mg and about 1000 mg, in single or divided doses. In managing the patient, the therapy should be initiated at a lower dose, perhaps about 1 mg to about 200 mg for a 70 kg person, and increased up to about 1000 mg or higher depending on the patient""s global response. It is further recommended that infants, children, patients over 65 years, and those with impaired renal or hepatic function, initially receive low doses, and that they be titrated based on individual response(s) and blood level(s). It may be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art. Further, it is noted that the clinician or treating physician will know how and when to interrupt, adjust or terminate therapy in conjunction with individual patient response. The terms xe2x80x9ctherapeutic amountxe2x80x9d and xe2x80x9ctherapeutically effective amountxe2x80x9d are encompassed by the above-described dosage amounts and dose frequency schedules.
The instant invention is not bound by any theory of operation. The following discussion is provided for the benefit of those skilled in the art, and does not limit the scope of the claims.
Preferred pharmaceutical compositions are particular useful for the treatment of bacterial infections. Bacteria sense their population density through a phenomenon called quorum-sensing, in which the bacteria synthesize and secrete low molecular weight compounds into the surrounding medium. By detecting the concentration of these signaling compounds in the medium they sense their population density, and express different genes in response to it.
Quorum-sensing controls not only light production in bioluminescent marine bacteria, where it was first discovered, but also production of exotoxins and other virulence factors in pathogenic bacteria. In many cases, modern drug design relies upon designing compounds that interact with targeted cellular constituents, such as enzymes, receptors, and periplasmic binding proteins, to block or otherwise alter the interaction between the target and the naturally-occurring compound it binds in vivo, the signaling compound in the case of quorum-sensing. Consequently, many strategies target the structure of both the signaling compound and its receptor as a way of designing agents that disrupt this interaction, for example, by competing with the signaling compound for the binding site on the receptor.
Recent work devoted to identifying a particularly important signaling compound known as autoinducer-2 (Al-2) is described in WO 00/32152, the disclosure of which is incorporated by reference in its entirety. AI-2 is believed to control quorum sensing at least in part by interacting with LuxP (SEQ ID NO: 1), a periplasmic binding protein from Vibrio harveyi that is involved with quorum-sensing in this bacterium. Crystallographic work on a luxP-AI-2 co-crystal, which resulted from LuxP expressed by recombinant Escherichia coli in the presence of biologically-produced AI-2, yielded the following structure (hereinafter called xe2x80x9cAI-2-boratexe2x80x9d) in which AI-2 (in the hydrated, gem-diol form of the keto group) binds at least in part to LuxP through the intermediacy of another species. 
This intermediary species was initially believed to be a carbonate but subsequently recognized to be a borate moiety, with the borate possibly arising from adventitious borate derived from borosilicate glass used in the experimental work. See X. Chen, S. Schauder, N. Potier, A. Van Dorsselaer, I. Pelczer, B. Bassler, and F. Hughson, xe2x80x9cStructural Identification of a Bacterial Quorum-Sensing Signal Containing Boron,xe2x80x9d Nature, Vol. 415, pp. 545-549 (2002), which is hereby incorporated by reference in its entirety.
The interaction between the borate ester of 4,5-dihydroxy-5-methyl-3(2H)-furanone (in its hydrated form) and LuxP indicates that borate ester formation may have biological relevance to the functioning of autoinducer-2. Borate ester formation may be a specific example of a general characteristic of autoinducer-2xe2x80x2s biological function, namely, as a chelating group for oxoanions such as borate, but including other oxoanions such as phosphate and sulfate. In nature, autoinducer-2 may function to present oxoanions to the autoinducer-2 receptor, with the oxoanion bound to autoinducer-2 either as a cyclic ester, as in the case of boron, or other structure.
Moreover, recognition of the role of 4,5-dihydroxy-5-methyl-3(2H)-furanone as a way of presenting oxoanions to a receptor may explain, at least in part, the activity of quorum-sensing inhibitors that incorporate a similar spatial disposition of functional groups. Thus, for example, preferred compounds as described herein having one or more heteroatoms in the five-membered ring, and therefore having similar hydrogen-bonding abilities to that found for the borate ester of 4,5-dihydroxy-5-methyl-3(2H)-furanone, have utility as competitive inhibitors of autoinducer-2 receptors, and therefore as therapeutic agents for controlling virulence by bacterial pathogens.